Preparation of Well-Defined Fluorescent Nanoplastic Particles by Confined Impinging Jet Mixing.

Research on the origin, distribution, detection, identification, and quantification of polymer nanoparticles (NPs) in the environment and their possible impact on animal and human health is surging. For different types of studies in this field, well-defined reference materials or mimics are needed. While isolated reports on the preparation of such materials are available, a simple and broadly applicable method that allows for the production of different NP types with well-defined, tailorable characteristics is still missing. Here, we demonstrate that a confined impinging jet mixing process can be used to prepare colloidally stable NPs based on polystyrene, polyethylene, polypropylene, and poly(ethylene terephthalate) with diameters below < 100 nm. Different fluorophores were incorporated into the NPs, to allow their detection in complex environments. To demonstrate their utility and detectability, fluorescent NPs were exposed to J774A.1 macrophages and visualized using laser scanning microscopy. Furthermore, we modified the NPs in a postfabrication process and changed their shape from spherical to heterogeneous geometries, in order to mimic environmentally relevant morphologies. The methodology used here should be readily applicable to other polymers and payloads and thus a broad range of NPs that enable studies of their behavior, uptake, translocation, and biological end points in different systems.

Understanding the Development, Standardization, and Validation Process of Alternative In Vitro Test Methods for Regulatory Approval from a Researcher Perspective.

Due to economic, practical, ethical, and scientific reasons, researchers, among others, are pushing for alternative in vitro test methods to replace or reduce existing animal experiments. In order for these tests to be more broadly used by the industrial sector and regulatory bodies, orchestrated efforts are required to show the robustness and reliability of in vitro methods, which can accelerate the use for early screening testing. Another way of increasing the use of alternatives is to coordinate validation studies, that is, multi-laboratory trials, and to gain regulatory approval and instatement as test guidelines or standard method. However, awareness of the exact standardization, validation, and approval process has been a major obstacle for many researchers. Herein, the process has been broken down into three main phases: i) test method development; ii) intra- and inter-laboratory validation; and iii) regulatory acceptance. This general process applies to all alternative methods seeking validation and approval, although the intricacies of different toxicological endpoints and/or chemical sectors may lead to additional work, particularly in the validation stage. The authors' aim is to provide insight in the development process of alternative methods with a focus on in vitro cell culture methods over validation to regulatory acceptance.

A novel 3D intestine barrier model to study the immune response upon exposure to microplastics.

The plausibility of human exposure to microplastics has increased within the last years. Microplastics have been found in different food types including seafood, salt, sugar and beverages. So far, human health effects of microplastics after ingestion are unknown. Herein, we designed a novel, three-dimensional in vitro intestinal model consisting of the human intestinal epithelial cell lines Caco-2 and HT29-MTX-E12 as well as human blood monocyte-derived macrophages and dendritic cells that is suitable to assess the possible effects of ingested microplastics. Relevant microplastic particles (in the order of 50-500 µm), including polymers representing tire wear and polyolefins, which represent major sources of microplastic in the EU, were compared to other polymer classes and an inorganic microparticle, healing earth, which is intended for human consumption. Microplastic particles were exposed at concentrations of 823.5-1380.0 µg/cm2 to the model using a dry powder insufflator system to aerosolize the particles directly on the intestinal model's surface. Cytotoxicity was investigated after 6, 24 and 48 h of exposure via measuring the release of lactate dehydrogenase. Inflammatory end points including the cytokines IL-8, TNFα and IL-1ß as well as changes of the barrier integrity after exposure were additionally monitored. We demonstrated that all of the microplastics and the healing earth particles did not cause any significant cytotoxicity or release of (pro-)inflammatory cytokines and did not change the barrier integrity of the co-culture at any of the time points investigated.

Emergence of Nanoplastic in the Environment and Possible Impact on Human Health.

On account of environmental concerns, the fate and adverse effects of plastics have attracted considerable interest in the past few years. Recent studies have indicated the potential for fragmentation of plastic materials into nanoparticles, i.e., "nanoplastics," and their possible accumulation in the environment. Nanoparticles can show markedly different chemical and physical properties than their bulk material form. Therefore possible risks and hazards to the environment need to be considered and addressed. However, the fate and effect of nanoplastics in the (aquatic) environment has so far been little explored. In this review, we aim to provide an overview of the literature on this emerging topic, with an emphasis on the reported impacts of nanoplastics on human health, including the challenges involved in detecting plastics in a biological environment. We first discuss the possible sources of nanoplastics and their fates and effects in the environment and then describe the possible entry routes of these particles into the human body, as well as their uptake mechanisms at the cellular level. Since the potential risks of environmental nanoplastics to humans have not yet been extensively studied, we focus on studies demonstrating cell responses induced by polystyrene nanoparticles. In particular, the influence of particle size and surface chemistry are discussed, in order to understand the possible risks of nanoplastics for humans and provide recommendations for future studies.

Efficient Receptor Mediated siRNA Delivery in Vitro by Folic Acid Targeted Pentablock Copolymer-Based Micelleplexes.

Novel, biocompatible polyplexes, based on the combination of cationic pentablock copolymers with folic acid functionalized copolymers, were designed and developed for target-specific siRNA delivery. The resulting micelleplexes spontaneously formed polymeric micelles with a hydrophobic core surrounded directly by a cationic poly-2-(4-aminobutyl)-oxazole (PABOXA) and subsequently shielded by hydrophilic poly-2-methyl-oxazole (PMOXA) layer. The described micelleplexes form highly stable particles even in complete serum after 24 h compared with the highly cationic polymer PEI, which show aggregate formation in serum containing buffer solution. Targeted siRNA delivery and gene knockdown could be shown using green fluorescent protein (GFP) expressing HeLa cells, resulting in ∼31% and ∼8% suppression of the expression of GFP for targeted and nontargeted micelleplexes, respectively. Comparison studies of folic-receptor positive HeLa cells with normal folic-receptor-negative HEK293 cells revealed involvement of receptor mediated cellular uptake of fluorescently labeled siRNA. The new designed nanocarrier showed no cytotoxicity, having a potential application. The presented concept of shielding a nucleic-acid complexing cationic chains with a stealth layer and combining it with receptor ligand overcomes typical problems with undesired protein and cell interactions in delivery of nucleic acids using polymeric systems, opening new doors for application if RNA inhibition in the organism.

A comparison of plasmid DNA delivery efficiency and cytotoxicity of two cationic diblock polyoxazoline copolymers.

Cationic polymers as non-viral gene delivery carriers are widely used because of their strong condensing properties and long-term safety, but acute cytotoxicity is a persistent challenge. In this study, two types of polyplexes were prepared by co-formulating plasmid DNA and two cationic diblock copolymers PABOXA5-b-PMOXA33-PA (primary amine) and PABOXA5-b-PMOXA33-TA (tertiary amine) to check their transfection efficacies in HeLa cells and HEK293T cells, respectively. The plasmid DNA/PABOXA5-b-PMOXA33-PA polyplex showed higher transfection efficacy compared to the plasmid DNA/PABOXA5-b-PMOXA33-TA polyplex under an N/P ratio of 40. Both polymers exhibited low toxicity, attributed to the shielding effect of a hydrophilic, noncharged block. Mechanistic insight into differential transfection efficiencies of the polymers were gained by visualization and comparison of the condensates via transmission electron and atomic force microscopy. The results provide information suited for further structure optimization of polymers that are aimed for targeted gene delivery.

Intelligent nanomaterials for medicine: carrier platforms and targeting strategies in the context of clinical application.

Nanomedical approaches are a major transforming factor in medical diagnosis and therapies. Based on important earlier work in the field of liposomal drug delivery and metallic nanomaterials, the last decade has brought a broad array of new and improved intelligent nanoscale platforms which are not only suited to deliver drugs and imaging agents but also to carry advanced functionality including internal and external stimuli-responsiveness in a highly targeted fashion to a diseased area. This review focuses on required properties and differences of basic delivery platforms in regard to deliver smart functionality, on building blocks suited to enhance tissue-, cell- and receptor-specific targeting and on nano-bio interaction. Further it discusses advantages and disadvantages of those platforms for future clinical application with regard to the subject of complement activation and hypersensitivity reactions in particular against polyethylene glycol (PEG) and possible functionalization with nanosize switches. FROM THE CLINICAL EDITOR: This review focuses on the properties of platforms designed to deliver smart functionality, using appropriate building blocks to enhance tissue-, cell-, and receptor-specific targeting. The authors also discuss potential complications such as complement activation and hypersensitivity reactions, and possible functionalization with nanosize switches.

Histomorphometric, CT arthrographic, and biomechanical mapping of the human ankle.

BACKGROUND: The specific morphological and biomechanical characteristics of the osteochondral unit of the ankle joint are not yet fully understood. This anatomical study aimed to map regional thickness of the articular hyaline uncalcified cartilage and its adjacent layers of mineralized cartilage and subchondral bone as well as to measure the regional indentation stiffness of human ankle joint cartilage. MATERIALS AND METHODS: A total of 20 pairs of human cadaver ankle joints (median age: 78 years) were evaluated by histomorphometry and multidetector row double-contrast CT arthrography for cartilage thickness in 17 distinct anatomical regions. In addition, regional distribution of the subchondral bone plate and of the mineralized cartilage was scrutinized histologically. Cartilage indentation stiffness was measured using an arthroscopic handheld device (Artscan200), especially validated for use in thin cartilage. The correlation between the thickness of different components of the osteochondral unit and the cartilage indentation stiffness was evaluated. RESULTS: The thinnest uncalcified cartilage was measured at the anterior talar dome and the distal fibula. The thickest uncalcified cartilage was found in the mid and posterior talar dome, as well as in the tibial plafond. Mineralized cartilage and subchondral bone showed highest values at the anteromedial talar dome. Cartilage indentation stiffness showed a bicentric distribution pattern in 14/20 ankle pairs and was highest in regions with thin cartilage. Positive correlation between the thickness of the mineralized cartilage and the subchondral bone plate was found. No correlation between the thickness of the uncalcified and the mineralized cartilage could be identified. CONCLUSION: This anatomical study provides a comprehensive mapping of the osteochondral unit of the human ankle joint in elderly people. Articular hyaline uncalcified cartilage and the subchondral bone plate showed clear regional differences and were reciprocally distributed. Cartilage indentation stiffness was inversely correlated to cartilage thickness in elderly people. CLINICAL RELEVANCE: Thorough understanding of the osteochondral unit of the ankle joint could be helpful for clinicians and researchers in the development of improved operative repair techniques for osteochondral defects in the ankle joint, for example, in constructing specific tissue-engineered osteochondral plugs.

How small is the big problem? Small microplastics <300 µm abundant in marine surface waters of the Great Barrier Reef Marine Park.

Particle size plays an important role in determining the behaviour, fate and effects of microplastics (MPs), yet little is known about MPs <300 µm in aquatic environments. Therefore, we performed the first assessment of MPs in marine surface waters around the Whitsunday Islands region of the Great Barrier Reef Marine Park, Australia, to test for the presence of small MPs (50-300 µm) in-situ. Using a modified manta net, we demonstrate that MPs were present in all marine surface water samples, with a mean sea surface concentration of 0.23 ± 0.03 particles m-3. Microplastics were mainly blue, clear and black fibres and fragments, consisting of polyethylene terephthalate, high-density polyethylene and polypropylene plastic polymers. Tourism and marine recreation were considered the major contributing sources of MPs to surface waters around the Whitsunday Islands. Between 10 and 124 times the number of MPs exist in the 50 µm-300 µm size class, compared with the 1 mm-5 mm size range. This finding indicates that the global abundance of small MPs in marine surface waters is grossly underestimated and warrants further investigation. Research into the occurrence, characteristics and environmental fate of MPs <300 µm is needed to improve our understanding of the cumulative threats facing valuable ecosystems due to this smaller, potentially more hazardous size class.

Fate of engineered nanomaterials at the human epithelial lung tissue barrier in vitro after single and repeated exposures.

The understanding of the engineered nanomaterials (NMs) potential interaction with tissue barriers is important to predict their accumulation in cells. Herein, the fate, e.g., cellular uptake/adsorption at the cell membrane and translocation, of NMs with different physico-chemical properties across an A549 lung epithelial tissue barrier, cultured on permeable transwell inserts, were evaluated. We assessed the fate of five different NMs, known to be partially soluble, bio-persistent passive and bio-persistent active. Single exposure measurements using 100 µg/ml were performed for barium sulfate (BaSO4), cerium dioxide (CeO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NMs and non-nanosized crystalline silica (DQ12). Elemental distribution of the materials in different compartments was measured after 24 and 80 h, e.g., apical, apical wash, intracellular and basal, using inductively coupled plasma optical emission spectrometry. BaSO4, CeO2, and TiO2 were mainly detected in the apical and apical wash fraction, whereas for ZnO a significant fraction was detected in the basal compartment. For DQ12 the major fraction was found intracellularly. The content in the cellular fraction decreased from 24 to 80 h incubation for all materials. Repeated exposure measurements were performed exposing the cells on four subsequent days to 25 µg/ml. After 80 h BaSO4, CeO2, and TiO2 NMs were again mainly detected in the apical fraction, ZnO NMs in the apical and basal fraction, while for DQ12 a significant concentration was measured in the cell fraction. Interestingly the cellular fraction was in a similar range for both exposure scenarios with one exception, i.e., ZnO NMs, suggesting a potential different behavior for this material under single exposure and repeated exposure conditions. However, we observed for all the NMs, a decrease of the amount detected in the cellular fraction within time, indicating NMs loss by cell division, exocytosis and/or possible dissolution in lysosomes. Overall, the distribution of NMs in the compartments investigated depends on their composition, as for inert and stable NMs the major fraction was detected in the apical and apical wash fraction, whereas for partially soluble NMs apical and basal fractions were almost similar and DQ12 could mainly be found in the cellular fraction.

The micro-, submicron-, and nanoplastic hunt: A review of detection methods for plastic particles.

Plastic particle pollution has been shown to be almost completely ubiquitous within our surrounding environment. This ubiquity in combination with a variety of unique properties (e.g. density, hydrophobicity, surface functionalization, particle shape and size, transition temperatures, and mechanical properties) and the ever-increasing levels of plastic production and use has begun to garner heightened levels of interest within the scientific community. However, as a result of these properties, plastic particles are often reported to be challenging to study in complex (i.e. real) environments. Therefore, this review aims to summarize research generated on multiple facets of the micro- and nanoplastics field; ranging from size and shape definitions to detection and characterization techniques to generating reference particles; in order to provide a more complete understanding of the current strategies for the analysis of plastic particles. This information is then used to provide generalized recommendations for researchers to consider as they attempt to study plastics in analytically complex environments; including method validation using reference particles obtained via the presented creation methods, encouraging efforts towards method standardization through the reporting of all technical details utilized in a study, and providing analytical pathway recommendations depending upon the exact knowledge desired and samples being studied.

Spatial and temporal analysis of meso- and microplastic pollution in the Ligurian and Tyrrhenian Seas.

Areas like the Mediterranean coastlines, which have high population density, represent locations of high pollution risk for surrounding environments. Thus, this study aims to compare data on the abundance, size, and composition of buoyant plastic particles collected during two weeks in 2019 in the Ligurian and Tyrrhenian Seas with data from 2018. The results from 2019 show average meso- and microplastic particle concentrations of 255,865 ± 841,221 particles km-2, or 394.19 ± 760.87 g km-2; values which differ significantly from those reported in 2018. Microplastic particles accounted for 88.7% of the sample; the majority of which had a size range being between 1 and 2.5 mm. These data are an important milestone for long-term monitoring of the highly variable plastic pollution levels within this region; showing overlaps in zones of pollutant accumulation in addition to increased overall concentrations of plastic particles compared to previous data.

Publisher Correction: Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Subcellular Imaging of Liquid Silicone Coated-Intestinal Epithelial Cells.

Surface contamination and the formation of water bridge at the nanoscopic contact between an atomic force microscope tip and cell surface limits the maximum achievable spatial resolution on cells under ambient conditions. Structural information from fixed intestinal epithelial cell membrane is enhanced by fabricating a silicone liquid membrane that prevents ambient contaminants and accumulation of water at the interface between the cell membrane and the tip of an atomic force microscope. The clean and stable experimental platform permits the visualisation of the structure and orientation of microvilli present at the apical cell membrane under standard laboratory conditions together with registering subcellular details within a microvillus. The method developed here can be implemented for preserving and imaging contaminant-free morphology of fixed cells which is central for both fundamental studies in cell biology and in the emerging field of digital pathology.

Biodistribution, Clearance, and Long-Term Fate of Clinically Relevant Nanomaterials.

Realization of the immense potential of nanomaterials for biomedical applications will require a thorough understanding of how they interact with cells, tissues, and organs. There is evidence that, depending on their physicochemical properties and subsequent interactions, nanomaterials are indeed taken up by cells. However, the subsequent release and/or intracellular degradation of the materials, transfer to other cells, and/or translocation across tissue barriers are still poorly understood. The involvement of these cellular clearance mechanisms strongly influences the long-term fate of used nanomaterials, especially if one also considers repeated exposure. Several nanomaterials, such as liposomes and iron oxide, gold, or silica nanoparticles, are already approved by the American Food and Drug Administration for clinical trials; however, there is still a huge gap of knowledge concerning their fate in the body. Herein, clinically relevant nanomaterials, their possible modes of exposure, as well as the biological barriers they must overcome to be effective are reviewed. Furthermore, the biodistribution and kinetics of nanomaterials and their modes of clearance are discussed, knowledge of the long-term fates of a selection of nanomaterials is summarized, and the critical points that must be considered for future research are addressed.

Economic efficiency of gate-keeping compared with fee for service plans: a Swiss example.

STUDY OBJECTIVE: The impact of isolated gate-keeping on health care costs remains unclear. The aim of this study was to assess to what extent lower costs in a gate-keeping plan compared with a fee for service plan were attributable to more efficient resource management, or explained by risk selection. DESIGN: Year 2000 costs to the Swiss statutory sick funds and potentially relevant covariates were assessed retrospectively from beneficiaries participating in an observational study, their primary care physicians, and insurance companies. To adjust for case mix, two-part regression models of health care costs were fitted, consisting of logistic models of any costs occurring, and of generalised linear models of the amount of costs in persons with non-zero costs. Complementary data sources were used to identify selection effects. SETTING: A gate-keeping plan introduced in 1997 and a fee for service plan, in Aarau, Switzerland. PARTICIPANTS: Of each plan, 905 randomly selected adult beneficiaries were invited. The overall participation rate was 39%, but was unevenly distributed between plans. MAIN RESULTS: The characteristics of gate-keeping and fee for service beneficiaries were largely similar. Unadjusted total costs per person were Sw fr 231 (8%) lower in the gate-keeping group. After multivariate adjustment, the estimated cost savings achieved by replacing fee for service based health insurance with gate-keeping in the source population amounted to Sw fr 403-517 (15%-19%) per person. Some selection effects were detected but did not substantially influence this result. An impact of non-detected selection effects cannot be ruled out. CONCLUSIONS: This study hints at substantial cost savings through gate-keeping that are not attributable to mere risk selection.

Designing switchable nanosystems for medical application.

Within the last decade, nanotechnology has had a major impact on preclinical development in medicine, shaping the emerging scientific field of nanomedicine. Diverse nanomaterial platforms have been introduced as a carrier systems for the delivery of a variety of payloads (e.g. drugs, proteins, peptides, nucleic acids) but additional improvement by stimulus responsiveness would be of tremendous significance. The design of intelligent, stimuli responsive nanosystems promises to expand diagnostic and therapeutic options in medicine by making available an array of highly effective, well tolerated platforms that go beyond simple delivery of drugs or imaging agents. Controlled by internal triggers which may be characteristic for a disease or by external devices that permit tight spatiotemporal control of activity, enhancement of desired therapeutic effects and further suppression of side effects in remote organs may be possible. This review focuses on the toolbox of available internal and external switches suited for the integration into nanoscale carriers and on the clinical experience with stimuli-responsive nano-platforms. A substantial body of evidence shows that internal stimuli including pH, redox potential, enzymatic activity and temperature are suited to trigger nanosystems. For some such systems, clinical trials are in progress, but solid clinical proof of significant patient benefit will be required next. Externally controlled systems include electromagnetically-, temperature-, light-, radiation- and ultrasound triggered systems, and for certain clinical indications, such systems have already proven clinical benefit.

Polydimethylsiloxane embedded mouse aorta ex vivo perfusion model: proof-of-concept study focusing on atherosclerosis.

Existing mouse artery ex vivo perfusion models have utilized arteries such as carotid, uterine, and mesenteric arteries, but not the aorta. However, the aorta is the principal vessel analyzed for atherosclerosis studies in vivo. We have devised a mouse aorta ex vivo perfusion model that can bridge this gap. Aortas from apoE((-/-)) mice are embedded in a transparent, gas-permeable, and elastic polymer matrix [polydimethylsiloxane (PDMS)] and artificially perfused with cell culture medium under cell culture conditions. After 24 h of artificial ex vivo perfusion, no evidence of cellular apoptosis is detected. Utilizing a standard confocal microscope, it is possible to image specific receptor targeting of cells in atherosclerotic plaques during 24 h. Imaging motion artifacts are minimal due to the polymer matrix embedding. Re-embedding of the aorta enables tissue sectioning and immuno-histochemical analysis. The ex vivo data are validated by comparison with in vivo experiments. This model can save animal lives via production of multiple endpoints in a single experiment, is easy to apply, and enables straightforward comparability with pre-existing atherosclerosis in vivo data. It is suited to investigate atherosclerotic disease in particular and vascular biology in general.